1. Field of the Invention
The present invention relates to therapeutics and methods of treating cancer. In particular, the present invention relates to modified programmed-cell-death executioner genes.
2. Description of the Prior Art
DNA-degrading enzymes are important mediators of aptoptosis. DNase 1, for example, is a powerful DNA-degrading enzyme, that preferentially cleaves DNA at phosphodiester linkages adjacent to a pyrimidine nucleotide, producing tetranucleotides, and also binds actin. DNase 1 acts on both single- and double-stranded DNA as well as chromatin. DNase 1 is the major extracellular DNase protein and a major executioner of intracellular apoptosis, i.e. cell death (Peitsch, et al., EMBO J. 1993, 12:371-7; 1993; Boone, et al., Biol Reprod 1997, 57:813-21). In its native form, DNase 1 cannot trigger intracellular apoptosis. Peitsch, et al. proposed that processes that initiate apoptosis allow the rapid access of endoplasmic reticulum enzymes to the nucleus, resulting in the apparent “activation” of DNase 1, and COS cells transfected with cDNA of rat parotid DNase 1 resulted in DNA fragmentation. The signal peptide, which diverts the protein away from the nucleus, was not removed. A nuclear localization signal was not included; therefore, DNase 1 did not localize to the nucleus. The actin binding site was not mutated, and therefore, the DNase 1 was not resistant to inhibition by actin.
Normal and cancerous cells protect themselves from premature death by the powerful DNA-digestive DNase 1 by four main mechanisms: (i) the nucleus of cells is enveloped by a nuclear membrane that is impenetrable to DNase 1; (ii) the DNase 1 protein is lacking a nuclear localization signal (NLS), which other DNases such as DNase γ have (Lechardeur, et al., J Cell Biol 2000, 150:321-4; Shiokawa, et al., Biochem J 2003, 376:377-81), and the NLS is needed to direct a protein after translation back into the nucleus; (iii) the DNase 1 protein is equipped with a signal peptide (SP), which directs it away from the nucleus, to the endoplasmic reticulum, where it is packaged in an endosome to contain its activity and to either be secreted from the cell or to reside in small amounts around the nucleus; and (iv) even if DNase 1 could leak into the nucleus, it would be inactivated by nuclear actin. Thus, DNase 1 could have access to the cell's DNA only after the activated apoptosis cascade led to initial disintegration of the nucleus membrane. Various combinations of these four mechanisms likely protect normal and cancerous cells from additional DNA-degrading enzymes to DNAse I.
Saito, et al. (J Neuro-Oncol 2003, 63:25-31) induced DNA fragmentation by transfecting human glioma cells with DNase γ. In the years since this publication, neither this group nor any other group has pursued therapeutic application for DNase γ. Progress was made only in showing histological evidence in rodent liver that DNase γ has a role in Fas-independent apoptotic DNA fragmentation (Higami, et al., Cell Tissue Res 2004, 316: 403-7), and in further biochemical characterization of DNase γ (Sunaga, et al., Bioorg Med. Chem. 2006, 14:4217-26; Mizuta, et al., Biochem Biophys Res Commun. 2006, 345:560-7). Caspase-activated DNase (CAD, DFF40) has been used by Ben-Yehuda, et al. (Clin Cancer Res 2003, 9:1179-90) in the form of a chimeric protein with Gonadotropin-releasing hormone (GnRH-DFF40) to induce apoptosis in a colon adeno-carcinoma cell line. In the years since that publication, this group did not disclose any further advances toward therapy with their approach. Progress was made only to show that the GnRH-based chimeric protein can be used as a tool for the detection of adenocarcinoma (Lichtenstein, et al., Int J Oncol 2005, 27:143-8).
In 1993, Polzar, et al. (Eur J Cell Biol 1993, 62:397-405) demonstrated that overexpression of native DNase 1 caused apoptosis of COS-cells. In the same year, the same group (Petisch, et al., EMBO J. 1993, 12:371-7) reported that overexpression of DNase 1 alone cannot induce apoptosis because it has no access to the nucleus. Napirei, et al. (Biochem J 2005, 389: 355-64) also overexpressed native DNase 1 and observed neither apoptosis nor localization of DNase 1 in the nucleus. Rat DNase 1 and murine DNase 1l3 were compared, and neither contained an NLS, and the actin binding site was not mutated. Oliveri, et al. (Eur J Immunol 2001, 31:743-51) accelerated apoptosis 2-4 fold by adding chemotherapeutic drugs to cells, which stably overexpressed the DNase 1 gene. The signal peptide-DNase 1 by itself has not been shown to induce apoptosis. In Oliveri, et al., the signal peptide was not removed, an NLS was not included, and the actin binding site was not mutated, and therefore the DNase 1 did not localize to the nucleus and it was not actin resistant.
Napirei, et al. (Biochem J 2005, 389:355-64) deleted the signal peptide from DNase 1 and observed localization of DNase 1 in both the cytoplasm and nucleus, but no apoptosis.
Linardou, et al. (Int J Cancer, 2000, 86:561-9) created a scFv-DNase 1 chimera against human placental alkaline phosphatase (hPLAP) that decreased cell viability when measuring the level of DNA synthesis in a tritium-labeled thymidine (3H-TdR) incorporation assay. Bovine DNase 1 was used. The signal peptide was not removed, and an NLS was not included. The actin binding site was not mutated, and therefore, the chimera was not resistant to DNase 1 inhibition by actin. Apoptosis tests were not performed, only cytotoxicity was tested. The scFv-Ab seemed to be cytotoxic as well, suggesting necrosis in addition to possible apoptosis.
The actin binding site on DNase 1 has been inactivated to increase the potency of DNase 1 to dilute viscosity of upper respiratory secretions in Cystic Fibrosis (CF) patients (Ulmer, et al., PNAS 1996, 93:8225-9; Pan, et al., J Biol Chem 1998, 273:18374-81) and for treatment of systemic lupus erythematosus (Pan, et al., J Biol Chem 1998, 273:18374-81). Recombinant human DNase 1 (PULMOZYME®, Genentech) was approved by the FDA in December 1998 as an aerosolized mucolytic agent for the management of patients with CF. This shows that DNase 1 is safe to administer to humans. Ulmer, et al. showed that the binding site for actin was mutated and increased DNAse 1's ability to digest DNA in sputum, thus, reducing its viscosity. The DNase 1 was not aimed at functioning inside cells or inducing apoptosis in cells. The signal peptide, which diverts the protein away from the nucleus, was not removed, and an NLS was not included.
None of the above described modifications of DNase 1 have been shown to be successful in treating cancer. There remains a need for a method of using DNases, and especially DNase 1, as therapeutics for cancer.
Malignant melanoma is the most aggressive form of cancer. Its dimensions are reported in millimeters. Tumor thickness approaching 4 mm presents a high risk of metastasis. A diagnosis of metastatic melanoma carries with it a median survival of 6-9 months. At the time of diagnosis, 20% of patients have metastasis; 16% to the lymph nodes and 4% to the distal organs. Current methods of treatment include surgical resection, radiation, chemotherapy with dacarbazine, tamoxifen, or temozolamide, or immunotherapeutics such as IL-2, IFN-α. Only a minority of patients respond to these methods of treatment. There is a general lack of efficacy and a high rate of severe side effects experienced by those patients using non-surgical treatments. The trend of therapeutics is going away from broad-spectrum cytotoxic chemotherapy and towards specific molecularly targeted therapies. Therefore, more effective treatments are needed for malignant melanoma.
Some of the therapeutics that have been developed as specific molecularly targeted therapies include immunotherapies such as vaccines (e.g. Hi-8™ MEL-vaccine (Oxxon Therapeutics)) and ONCOVEX® (BioVex, Inc.), as well as biotherapeutics that perform functions such as targeting anti-apoptosis/resistance (e.g. Bcl2, IAP, NF-KB inhibition), promoting apoptosis (e.g. TRAIL ligands), siRNA (e.g. against Mitf), and suicide gene-therapy (e.g. HSV-tk).
Gene therapy, i.e. the transfer of genetic material into living cells for treatment or prevention of disease, has grown in popularity for use as a treatment over the years. The United States conducts the most gene therapy clinical trials, with some conducted in Europe and Asia. The United States also leads the rest of the world in the study of malignant melanoma with gene therapy clinical trials.
Gene therapy targeted to melanoma cells involves the introduction of “suicide” genes, such as a herpes simplex virus thymidine kinase gene (HSVtk), the transfer of tumor suppressor genes such as p53 gene or p16INK4a, the inactivation of oncogenic signaling pathways such as ras, c-myc, and signal transducers and activators of transcription-3 (Stat3), and the introduction of genes encoding immunologically relevant molecules such as allogenic MHC class I genes, cytokine genes (GM-CSF, IL-2, IFNs), and co-stimulatory molecules (B7.1). Gene therapy can also be targeted to the host's immune cells. T cells are targeted with the introduction of neomycin phosphotransferase gene and chimeric receptor (IL-2R/GM-CSFR). Dendritic cells are targeted with genes encoding melanoma antigens (MART-1/Melan A) and CD40 ligand.
Several clinical trials involving gene therapy are currently being conducted. GLYBERA® (alipogene tiparvovec, Amsterdam Molecular Therapeutics) is an AAV-1 vector encoding lipoprotein lipase for treatment of patients with lipoprotein lipase deficiency. INGN-241 (Introgen) is an E1-deleted, replication-incompetent adenoviral vector encoding melanoma-differentiation-associated gene-7 (mda-7; interleukin-24) for treatment of metastatic melanoma. TNFerade (GenVec) is an E1-, E3-, and E4-delected andenoviral vector encoding human TNF-α under the control of the radiation-inducible early growth response promoter for use in pancreatic cancer. A retrovirus by MolMed encoding herpes simplex virus thymidine kinase transduced ex vivo into hematopoietic stem cells is used for graft-versus-host disease. ALLOVECTIN-7® velimogene aliplasmid, Vical) is a DNA plasmid encoding the human leukocyte antigen-B7 (HLA-B7) and β2-microglobulin complex in the context of cationic lipid mixture (DMRIE/DOPE) for use in chemotherapy-naïve patients with metastatic melanoma. PROSAVIN® (Oxford Biomedica) is combined lentivirus and equine infectious anemia virus vectors encoding aromatic amino acid decarboxylase, tyrosine hyroxylase and GTP-cyclohydrolase-1 used for Parkinson's disease. tgAAC-94 (Targeted Genetics) is an AAV-2 encoding IgG1Fc and the TNF-α receptor used in rheumatoid arthritis.
There are also several current gene therapies in clinical trials directed at targeting apoptosis in cancer. Anti-TRAILR1 agonistic antibody (Human Genome Sciences/Cambridge Antibody Technology/Takeda), Anti-TRAILR2 agonistic antibody (Human Genome Sciences/Cambridge Antibody Technology), and TRAIL (Genentech/Amgen) are used for solid tumors. GENASENSE® (oblimersen, antisense oligonucleotide targeting BCL2, Genta, Inc.) was used in malignant melanoma, multiple myeloma, and chronic lymphocytic leukemia (CLL). SPC-2996 (antisense oligonucleotide targeting BCL2, Santaris Pharma), AT 101 ((−)-Gossypol, Ascenta Therapeutics Inc.), and small molecule BCL2-family inhibitor (Gemin X Biotech) are used for CLL. Other apoptosis-inducing agents include ABT-737 (small molecule BCL2-family inhibitor, Abbott Laboratories/Idun Pharmaceuticals), IPI-983L/IPI-194 (small molecule BCL2-family inhibitors, Infinity Pharmaceuticals), XIAP-BIR2 inhibitor (Burnham Institute), XIAP-BIR3 inhibitor (UT Southwestern), XIAP-BIR3 inhibitor (Abbott Laboratories), and Nutlins (MDM2 inhibitors, Wyeth).
Several gene therapies have been developed to specifically target apoptosis in melanoma by sensitizing melanoma cells to TRAIL-mediated apoptosis. Some agents upregulate TRAIL receptors to cause DNA damage and upregulation of apoptosis through p53 (cisplatin/doxorubicin, betulinic acid, CD347 retinoid, TNF-α). Other agents potentiate the mitochondrial pathway and decrease BCL2, BCL-XL, and MCL-1 (Antisense to BCL2, BCL-XL, and MCL-1), downregulate BCL2, BCL-XL, and MCL-1 (MEK1 inhibitors, PD98059), damage mitochondria (cisplatin/doxorubicin), and increase apoptosis activating factor (APAF) levels by inhibiting methylation (5-aza-deoxycytidine). Agents inhibit NF-κB activation by proteasome inhibition and upregulation of IκBα (PS341), and competing with p53 for co-transcriptional factors (temozolomide and vinblastine). Also, agents downregulate inhibitor of apoptosis protein (IAP) levels by decreasing XIAP levels (actinomycin-D, fludarabine, second mitochondria-derived activator of caspase (SMAC)/DIABLO constructs).
Apoptosis is a physiological mechanism of programmed cell death by which unwanted cells are eliminated from tissues in response to specific stimuli. The apoptosis cascade is shown in FIG. 1. The current failure of the above described gene therapy treatments results from the fact that the majority of cancers protect themselves by inactivating or underexpressing death receptors, not expressing or inactivating intermediate components of the apoptosis cascade, and overexpressing anti-apoptotic proteins that inactivate pro-apoptotic components along the apoptosis cascade as shown in FIG. 2. Therefore, a method of treatment is needed that overcomes the difficulties presented by the natural protection mechanisms of cancer cells against the apoptosis cascade.